2.4. Cell-Based Assays

The human AML cell lines, OCI-AML3 (carrying the NPM1 gene mutation A and the DNMT3A^R882C mutation), IMS-M2 (carrying the NPM1 gene mutation A), OCI-AML2 (with the wild-type NPM1 and DNMT3A^R635W mutation), and SKM-1 and MOLM-13 (with the wild-type NPM1) were previously reported [37,38]. The cell lines were purchased from the Deutsche Sammelung von Mikroorganismen und Zellkulturen (DSMZ) (Braunschweig, Germany) cell bank and maintained according to the vendors’ instructions. Briefly, cultured cells were split every 3 days and maintained in an exponential growth phase. The cell lines were tested for mycoplasma using the Universal Mycoplasma Detection Kit (ATCC). The OCI-AML3 were grown in α-MEM supplemented with 20% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin (P/S), and all other cell lines were cultured in RPMI and supplemented with a 10–20% fetal bovine serum and 100 U/mL penicillin and 100 μg/mL streptomycin. The cells were kept at 37 °C in a 5% CO₂ incubator. Prior to screening, the appropriate cell concentration for all cell lines was determined. For the cell lines’ treatments, cells were maintained as follows: OCI-AML3 at 0.25 × 10⁶/mL, OCI-AML2 and IMS-M2 at 0.35 × 10⁶/mL, and SKM-1 and MOLM-13 at 0.5 × 10⁶/mL. The compounds were re-suspended at 10 mM stock in DMSO. Further dilutions were performed in phosphate-buffer-saline (PBS) for pharmacological experiments. The cells were exposed to compounds at different concentrations for the indicated time points and sampled as described below. Briefly, the cells (4 × 10⁵/mL) were seeded in 384-well plate and treated for 48 h with various concentrations (0.001 μM to 100 μM) of either one of the compounds, T101, T126, and T159, or NSC348884, as a positive control [39]. Each plate included DMSO at 0.1% as a negative control. The metabolic cell activity was measured by using the CellTiter Blue proliferation agent (Roche) as described in the manufacturer’s procedure. Briefly, CellTiter Blue solution (Resazaurin) was added to each well and the plates were incubated at 37 °C with 5% CO₂ for 4 h. During the incubation time, the resazurin is reduced to fluorescent resorufin in metabolically and proliferating living cells. The fluorescence intensity was measured using the automated Tecan plate reader (Tecan Group Ltd., Männedorf, Switzerland) at an excitation/emission wavelength of 531/572 nm. The fluorescence readouts were reported as relative fluorescence units (RFU) and read on a luminescence plate reader (Infinite 200 Pro, Tecan, CA, USA). The dose response curves (maximal inhibitory concentration (IC₅₀)) were obtained by non-linear regression analysis using the GraphPad Prism 5.0 program. The concentration is shown as a logarithmic function. All the experiments were performed at least in triplicate, with technical repetition. The results are expressed as mean ± standard error of the mean (SEM). For the Colony Forming Unit (CFU) assay, 10 × 10⁶ CD34+ human hematopoietic progenitors were received from unique healthy donors, upon signed informed consent, and were cultured in SFEM II (Serum-free medium) and P/S at a density of 1 × 10⁶/mL with cytokines that are important for cell division and self-renewal: fms-like tyrosine kinase 3 (FLT3)-ligand, FLT3L (100 ng/mL), Stem Cell Factor, SCF (100 ng/mL) and Trombopoietin, and TPO (50 ng/mL) for 48 h before plating them on a Methocult medium.

For the CFU assay, 500 CD34+ human cells were plated in a MethoCult™ enriched medium (see Table S4) on a 6-well plate (untreated cells versus T126 at 1, 2.5, and 5 µM, each condition in triplicate). The plates were then incubated for 14 days at 37 °C (5% CO₂), and the images were captured and the colonies counted by STEMvision™. STEMvision™ is a bench-top instrument and computer system designed specifically for the automated imaging and counting of hematopoietic colonies in the CFU assay. This system has been optimized for use with MethoCult™ media and meniscus-free SmartDish™ culture ware. The use of this standardized platform significantly improves the accuracy and reproducibility of the human CFU assay. Mobilized peripheral blood (MPB) analysis packages have been used to accurately count colonies.

Immunoblotting was conducted as described previously [37]. Briefly, fresh cell pellets (0.2–0.5 × 10⁶/test for AML cell lines) were dissolved directly in 30–60 μL of Laemmli sample buffer 1× (1.5 M Tris-HCl pH 6.8, glycerol, β-mercaptoethanol, SDS, bromophenol blue) and boiled at 95 °C for 5 min. The proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on Precast 4–15% gradient gels (Biorad, Hercules, CA, USA) transferred onto nitrocellulose membranes (GE Healthcare, Piscataway, NJ, USA) and probed with specific primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies (GE Healthcare Lifesciences). The polypeptides were visualized using enhanced chemiluminescence (Luminata Crescendo, Merck Millipore, Burlington, MA, USA) according to the manufacturer’s instructions. The bands were visualized using the Biorad Chemidoc and the images were processed using the image lab software (Biorad) and Adobe Photoshop (Ps CC 2019). β-actin expression levels were used as a control for protein loading, as indicated.

The viable cell count was evaluated daily using the Invitrogen Countess automated cell counter (Invitrogen, Carlsbad, CA, USA) by trypan blue exclusion, and individual growth curves were generated from these data.

The cell apoptosis was assessed using Annexin V APC- or FITC-conjugated antibodies and counterstained with Propidium Iodide (PI) or 7AAD for the detection of necrotic cells (Becton Dickinson, Franklin Lakes, NJ, USA), according to the manufacturer’s protocol. The data acquisition and analysis were done with a FACS Canto II flow cytometer and data were acquired and analyzed using Diva software (Becton Dickinson, Franklin Lakes, NJ, USA).

The primary antibodies used for western blot analyses are the following: mouse monoclonal anti-human β-actin (Clone AC-15; dilution 1:5000) from Sigma Aldrich (St. Louis, MO, USA); rabbit monoclonal anti-human Cleaved poly (ADP-ribose) polymerase (c-PARP) (Clone Asp 214; dilution 1:1000); rabbit monoclonal phospho-Akt (Ser473) (clone D9E, dilution 1.500); rabbit polyclonal phospho-mTOR (Ser2448) (dilution 1:500); rabbit polyclonal phospho-4E-BP1 (Ser65) (dilution 1:500); rabbit monoclonal Akt1 (clone D9R8K, dilution 1:500); rabbit polyclonal 4E-BP1 (dilution 1:500); and rabbit polyclonal mTOR (dilution 1:500), all from Cell Signaling (Cell Signaling Technology, Inc., Danvers, MA, USA).

All the experiments were performed at least in triplicate, as indicated, with technical repetition when possible. The results are expressed as mean ± standard error of the mean (SEM). A one- or two-tailed paired Student t-test with a normal-based 95% CI was applied for statistical analysis, as indicated. Statistical significance was defined as p < 0.05.